Research Summary

Stem cells are pluripotent cells that give rise to all tissues of the body, including neurons and glia of the central nervous system. Genome-scale complex regulatory processes unfold over time and space in the prenatal central nervous system to establish developmental programs that govern the differentiation of countless neuronal and glial cell types that populate the brain. Cell-intrinsic, species-specific genetic blueprints as well as cell-to-cell contacts and signaling systems govern these complex regulatory processes and render them more robust to perturbations. We hypothesize that slight deviations from typical programs of differentiation and patterning of neural stem cells predispose to disorders such as autism, Tourette syndrome, and schizophrenia. Induced pluripotent cells (iPSC) derived from living individuals and differentiated into telencephalic organoids recapitulate forebrain development in a dish, allowing us to examine typical and atypical development in patients with neuropsychiatric disorders and the underlying gene regulatory processes. Specifically we study the impact of noncoding RNAs, histone modifications, enhancer activity, and somatic mutations on typical and atypical brain development in human brain organoid culture and, whenever possible, in vivo.

Extensive Research Description

The Vaccarino laboratory has elucidated crucial mechanisms that regulate neural stem cell self-renewal, survival and differentiation. The lab has been examining conserved mechanisms of forebrain development using mouse models, and human-specific mechanisms by examining human stem and progenitor cells. Using induced pluripotent cells (iPSC) derived from living individuals the Vaccarino group is now examining how human stem cell differentiation varies across different genetic backgrounds, genders, and clinical phenotypes.

In the 1990’s Vaccarino and colleagues reported that an extracellular protein called Basic Fibroblast Growth Factor 2 (FGF2) increases the number of progenitors for excitatory cortical neurons in vitro (Vaccarino et al., 1995) and that a single microinjection of FGF2 into the cerebral ventricles of rat embryos doubled the number of excitatory pyramidal neurons generated during cortical development, producing an increase in cortical surface area (Vaccarino et al., 1999). Cortical pyramidal neurons form the primary scaffold of the cortex, and this was the first evidence that a single factor can elicit a permanent increase in pyramidal neuron number and cerebral cortical size in a mammalian species. Conversely, using knockout mice, Vaccarino and colleagues demonstrated that the Fgf2 gene product is essential for the generation of the species-specified number of excitatory neurons in the cerebral cortex by increasing cell proliferation in the cortical neuroepithelium (Vaccarino et al., 1999; Raballo et al., 2000; Korada et al., 2002). Subsequent work demonstrated that FGF receptors are essential for normal telencephalic development in region-specific fashion. The disruption of the Fgfr1 gene is sufficient to thwart cell proliferation within the hippocampal primordium, causing lifelong hippocampal atrophy (Ohkubo et al., 2004). However, abolishing the function of single FGF receptors causes minor abnormalities in cortex because all FGF receptors contribute redundantly to cortical development. However, the double knockout of Fgfr1 and Fgfr2 causes prefrontal cortex volume loss with fewer pyramidal cells (Stevens et al, 2010), and the combined knockout of Fgfr1, Fgfr2 and Fgfr3 in early neurogenesis depletes the cortical stem cell pool globally, resulting in premature ending of neurogenesis and decreased cortical surface area (Rash et al, 2011). Conversely, a microinjection of FGF2 in the lateral ventricles at pre-neurogenic stages of cortical development generates a massive enlargement of frontal cortical surface and the appearance of gyrus-like convolutions in stereotypic bilateral locations (Rash et al, 2013).

In summary, our studies have shown that FGFs establish the primary structure and the surface area of the cerebral cortex by promoting the self-renewal of neural stem cells and the differentiation of projection neurons from neuroepithelial precursors. We are currently studying the molecular mechanisms whereby FGF signaling affects the development of neocortical structure and size.

The lab has been redefining the roles of astroglial cells—extremely diverse cellular elements that evolve from a primary role of neural progenitors during embryonic development, to essential partners in neuronal migration, maturation, axon guidance. These cells have essential metabolic function in the adult brain and retain ability to divide and act as precursors. To further study the role of astroglial stem/progenitor cells in regenerative processes, the lab has generated the GFAP-CreERT2 transgenic line, in which the Cre recombinase can be transiently induced by a tamoxifen injection in GFAP+ astroglial cells (Ganat et al, 2006). By marking GFAP+ astroglial cells via recombination of genetic reporter we found that astrocytes can produce neurons in the immature brain, a process that is enhanced by hypoxic injury (Fagel et al., 2006, and Fagel et al, 2009; Bi et al, 2011) and dependent in part upon FGF receptor function (Fagel et al, 2009; Stevens et al, 2102). Indeed, Fgf ligands also play roles in different epochs of postnatal and adult life. FGF receptor functioning is required in astroglia to indirectly promote the maturation of inhibitory interneurons during the early postnatal development of the cerebral cortex (Müller Smith et al, 2014). Induced loss of FGF signaling only in postnatal astrocytes generates stereotypic locomotor hyperactivity and learning and memory defects that correlate with deficit in specific cell populations in the postnatal brain (Müller Smith et al, 2008, Stevens et al, 2012).

Another theme that the Vaccarino lab has been pursuing is whether an excitatory/inhibitory neuron imbalance in specific forebrain systems due to disparate etiologies (i.e., gene mutations; prenatal factors; environmental noxae) may predispose to neuropsychiatric disorders such as autism and Tourette syndrome.

For example, we have shown that individuals with Tourette’s syndrome (TS) have losses of Parvalbumin and cholinergic interneurons in specific regions of the striatum. TS is a developmental disorder of childhood characterized by motor and vocal tics. We demonstrated a large decrease in three classes of interneurons in the striatum of TS: Parvalbumin+; NOS+/NPY+/SST+; and cholinergic (Kalanithi et al, 2005; Kataoka et al, 2010). RNA sequencing of the postmortem striatum of deceased TS individuals revealed decreased expression of 308 genes, encompassing transcripts related to these three classes of interneurons, and decreased inhibitory neurotransmission in general. The study also revealed an up-regulation of 822 genes representing inflammatory response- and immune system-related genes (Lennington et al., 2014). The hypothesis is that altered postnatal maturation/survival of subsets of inhibitory neurons in cortico-basal ganglia circuits causes specific alterations in synchronous neuronal firing that may model disorders of the TS spectrum. Current studies aim at analyzing genetic and epigenetic differences in striatal and cortical tissue and within isolated cell types between TS and matched controls.

A deficit in inhibitory neurons has been found in the cortex of schizophrenic patients, and it has been postulated to occur in autism spectrum disorders (ASD). To model disorders of cortical development in a human system, the lab has recently adopted the induced pluripotent stem cell (iPSC) model and has developed a new way to derive cortical organoids from iPSCs as a window into normal and abnormal neuronal development (Mariani et al, 2012). Over the last several years Vaccarino and colleagues have derived hundreds of iPSC lines from patients with developmental disorders. They established a new protocol for converting iPSCs into brain organoids that reflect the human cerebral cortex at mid-fetal stages of development, which contain the main classes of excitatory cortical neurons and inhibitory interneurons. Using this tool, Vaccarino and colleagues have recently contributed fundamental work on neurodevelopmental alterations in severe, idiopathic ASD. Transcriptome and cell fate studies in organoids from ASD affected individuals as compared to unaffected family members indicated alterations in cell proliferation, overproduction of synapses and a striking increase in GABAergic neurons and their precursors, showing that ASD derived organoids have excess rather than defect in inhibitory GABAergic neurons (Mariani, Coppola et al, Cell, 2015). The work has also revealed an important role of the transcription factor FOXG1 in the overproduction of GABAegic precursor cells (Mariani, Coppola et al, Cell, 2015).

Ongoing studies integrate genomes, transcriptomes and cellular phenotypes to explore the etiology of autism and other neurodevelopmental alterations. As part of the PsychENCODE collaborative multi-site project, the lab will generate a genome-scale catalog of coding and noncoding RNAs and functional DNA elements in iPSC-derived organoids and human brain specimens of the same genetic background. Our object within the PsychENCODE is to verify to what extent the iPSC-derived organoids reflect true human brain development.

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